Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1998-09-14
2003-08-12
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S007400, C435S189000, C435S242000, C435S001100, C435S320100, C435S252100, C514S546000, C514S564000, C514S561000, C514S645000, C514S632000, C514S033000, C514S930000, C514S398000, C604S361000, C604S152000, C604S002000
Reexamination Certificate
active
06605447
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides methods and means for identifying and monitoring increased or decreased levels of inducible nitric oxide synthase in biological samples. Nitric oxide synthase activity is elevated in the inflammatory response resulting from several diseases or conditions, including, but not limited to, bacterial infections including urogenital tract infections and bacterially related sepsis, organ transplant rejection and cancer. Nitric oxide synthase activity is decreased in urine of patients with interstitial cystitis.
BACKGROUND OF THE INVENTION
One of the most common bacterial infections in humans is that of the urinary tract. Patients who need rapid diagnosis of urinary tract infections (UTIs) include premature newborn infants, prepubertal girls and young boys, sexually active women, elderly males and females, pre-operative patients, patients with chronic disease, patients with neurological disorders, patients with genitourinary congenital disorders including urethral valves and reflux, patients with sickle cell disease, patients with renal disease and polycystic kidney disease, patients having undergone renal transplantation and pregnant patients. The diagnosis of UTI in the elderly and in infants, in particular, is difficult because of different signs and symptoms and the inability to communicate, respectively. Failure to diagnose UTIs can lead to urosepsis, emphysematous cystitis and scarring.
Accordingly, there is a need for a rapid, cost effective, sensitive test for UTI. While a definitive diagnosis of urinary tract infections can be obtained by microbial culturing, this test is costly and results from the culture can take up to 48 hours to obtain. Newer technologies involving bacterial antibodies offer no clear advantages over culturing techniques. Studies have confirmed a direct relationship between urine nitrite and urinary tract infections. Accordingly, the Griess reagent which detects nitrite is commonly employed in a dipstick to screen urine for microorganisms. Leukocyte esterase dipstick tests are also used routinely for the rapid diagnosis of urinary tract infections (UTIs). However, these tests have been found to have different sensitivities and specificities for patients with different clinical manifestations. For example, Lachs et al. reported that this dipstick test for urinary tract infection was highly sensitive in patients with a high prior probability of infection but insensitive in patients with a low prior probability of infection (Lachs et al.,
Ann. Intern. Med.
1992, 117:135-40). Accordingly, as taught by Dr. Martin F. Shapiro in the commentary to this study, the dipstick test for urinary tract infection lacks sensitivity precisely in those patients for whom an effective diagnostic test would be most useful; patients with vague symptoms for whom the diagnosis is not clear. It has been estimated that the current dipstick technologies detect only approximately 50% of UTIs.
Diagnosis of transplant rejection is currently performed by evaluating symptoms along with changes in creatinine levels and by performing renal biopsies. Failure to diagnose acute rejection, which is the most common cause of loss of renal grafts, leads to longer hospital stays and higher medical costs.
Interstitial cystitis is a challenging medical problem because it is fundamentally a diagnosis of exclusion and because it is difficult to treat. Possible etiologies for interstitial cystitis include a viral or bacterial infection, lymphatic obstruction, deficiency of the bladder glycosaminoglycan layer, a pathologic substance in the urine, mast cell infiltration, or an alteration in the sensory nervous system (Batra et al.,
Interstitial Cystitis A.U.A. Update Series
1993, 12:1).
Nitric oxide synthase (NOS) is an enzyme found in a variety of mammalian tissues including neurons, macrophages and neutrophils. This enzyme converts L-arginine to nitric oxide and citrulline (Nathan, C.,
F.A.S.E.B. J.
1992, 6:3051). Isoforms of NOS have been characterized as Ca++ dependent and Ca++ independent enzymes in rodents (Nathan, C. and Xie, Q.,
Cell
1994, 78:915-918). In rodent macrophages and neutrophils, the Ca++ independent form, also referred to as inducible NOS (iNOS) produces large quantities of nitric oxide (NO) that can modulate immune, inflammatory and cardiovascular responses (Nathan, C. and Xie, Q.,
Cell
1994, 78:915-918 and Hibbs, J. B. Jr.,
Research Immunol.
1991, 142:565-569). In urine and plasma from rats, iNOS expression and elevated nitrite and nitrate levels are associated with rat cardiac and renal allograft rejection (Tanaka et al.,
Transplantation Proc.
1995, 27:576; Langrehr et al.,
J. Clin. Invest.
1992, 90:679; Cattell et al.,
Transplantation
1994, 58:1399; Yang et al.,
J. Clin. Invest.
1994, 94:714; Winlaw et al.,
Transplantation
1994, 58:1031; and Stevens et al.,
Transplant Proc.
1994, 58:1031). The Ca++ independent form is also activated as part of the host defense mechanism in humans and has been identified in the particulate fraction of the human urine pellet (Smith et al.,
Kidney Int.
1994, 45:586). Baseline human-urinary-particulate NOS activity is significantly higher in female urine as compared to male urine. Further, levels of this enzyme have been found to increase dramatically in urinary tract infections in both males and females. In addition, in human liver transplant patients, plasma nitrate (Ioannidis et al.,
Transplantation
1995, 59:1293) and plasma nitroso-compound levels (Devlin et al.,
Transplantation
1994, 58:592) have been found to increase with acute liver rejection. After allogeneic and autologous bone marrow transplantation in humans, there is also a significant rise of serum nitrate and nitrite levels preceding the onset of clinical symptoms of graft-versus-host disease (Weiss et al.,
Transplantation
1995, 60:1239). However, the presence of iNOS in human primary cells has been difficult to demonstrate.
Though much effort has been expended on the development of iNOS selective inhibitors as therapeutic agents (Jorens et al.,
Mediators of Inflamma.
1995, 4:75-89; Marletta, M. A.
J., Med. Chem.
1994, 37:1900-1907), the role of iNOS in human infectious and inflammatory processes is poorly understood. Despite some evidence suggesting the presence of iNOS in human macrophages, NOS in human neutrophils is not well characterized. Early reports indicate that human neutrophils can generate small amounts of NO (Wright et al.,
Biochem. Biophys. Res. Commun.
1989, 160:813-819) and may contain both a constitutive NOS (Bryant et al.,
Biochem. Biophys. Res. Commun.
1992, 189:558-564) and a NO sensitive guanylyl cyclase (Ney et al.,
Eiconsanoids
1990, 3:243-245 and Van Devort et al.,
J. Immunol.
1994, 152:4102-4108). More recent studies, however, have failed to detect either constitutive or inducible NOS activity (Yan et al.,
J. Immunol.
1994, 153:1825-1834). Highly enriched preparations of extravasated human polymorphonuclear leukocytes were reported to contain no message, protein or iNOS enzymatic activity (Miles et al.,
J. Leukoc. Biol.
1995, 58:616-622).
Accordingly, the role of iNOS in inflammatory and infectious organism mediated disease processes in humans and other mammals needs to be better determined. In the present invention, methods and means of detecting and monitoring iNOS levels and activity are provided. These methods have been found to be useful in identifying and monitoring increased or decreased levels of iNOS in biological samples which is useful in the diagnosis of diseases or conditions including, but not limited to, infections mediated by organisms such as bacteria, yeasts and viruses, sepsis, cancer, rheumatoid arthritis, transplant rejection and interstitial cystitis.
SUMMARY OF THE INVENTION
An object of the present invention is to provide methods of identifying and monitoring increased or decreased levels of inducible nitric oxide synthase and activity in biological samples. Means for detecting and monitoring levels
Sessa William C.
Smith Shannon D.
Weiss Robert M.
Wheeler Marcia A.
Licata & Tyrrell P.C.
Portner Ginny Allen
Smith Lynette R. F.
Yale University
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